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Copy-number estimation using Robust Multichip Analysis - Supplementary materials for the aroma.affymetrix lab session. Henrik Bengtsson & Terry Speed Dept of Statistics, UC Berkeley August 7, 2007 BioC 2007. Affymetrix chips. *. *. *. *. 5 µm. 5 µm.
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Copy-number estimation using Robust Multichip Analysis-Supplementary materials for the aroma.affymetrix lab session Henrik Bengtsson & Terry Speed Dept of Statistics, UC Berkeley August 7, 2007 BioC 2007
* * * * 5 µm 5 µm > 1 million identical 25bp sequences Generic Affymetrix chip * 1.28 cm 1.28 cm 6.5 million probes/chip Feature size: 100µm to 18µm to 11µmand now 5µm. Soon: 1µm, 0.8µm,with a huge increase in number of probes.
Abbreviated generic assay description • Start with target gDNA (genomic DNA) or mRNA. • Obtain labeled single-strandedtargetDNA fragments forhybridization to theprobeson the chip. • After hybridization, washing, staining and scanning we get a digital image. This is summarized across pixels to probe-level intensities before we begin. They are our raw data.
* * * * * * * * * * * 25 nucleotides Other DNA Other DNA Target seq. Other seq. X other PMs PM MM Affymetrix probe terminology Target DNA: ...CGTAGCCATCGGTAAGTACTCAATGATAG... ||||||||||||||||||||||||| Perfect match (PM): ATCGGTAGCCATTCATGAGTTACTA Mis-match (MM): ATCGGTAGCCATACATGAGTTACTA
Single Nucleotide Polymorphism (SNP) Definition: A sequence variation such that two chromosomes may differ by a single nucleotide (A, T, C, or G). Allele A:A ...CGTAGCCATCGGTA/GTACTCAATGATAG... Allele B:G A person is either AA, AB, or BB at this SNP.
* * * * * * * * * * * * * * * * * * * AA BB AB PMA >> PMB PMA << PMB PMA ¼PMB Probes for SNPs PMA:ATCGGTAGCCATTCATGAGTTACTA Allele A:...CGTAGCCATCGGTAAGTACTCAATGATAG...Allele B:...CGTAGCCATCGGTAGGTACTCAATGATAG...PMB:ATCGGTAGCCATCCATGAGTTACTA (Also MMs, but not in the newer chips, so we will not use these!)
Copy-number estimation using Robust Multichip Analysis (CRMA)
* * * * * * * * * * * * * * * * * * * AA BB AB PMA >> PMB PMA << PMB PMA ¼PMB Copy-number estimation using Robust Multichip Analysis (CRMA) Cross-hybridization: Allele A: TCGGTAAGTACTC Allele B: TCGGTATGTACTC
TT AT PMT AA + PMA offset Copy-number estimation using Robust Multichip Analysis (CRMA)
PMT PMA Copy-number estimation using Robust Multichip Analysis (CRMA)
Copy-number estimation using Robust Multichip Analysis (CRMA) Crosstalk calibration corrects for differences in distributions too log2 PM
Copy-number estimation using Robust Multichip Analysis (CRMA) Crosstalk calibration corrects for differences in distributions too log2 PM
* * * * * * * * * * * * * * * * * * PM = PMA +PMB Copy-number estimation using Robust Multichip Analysis (CRMA) AA AB PM =PMA+PMB * BB PM =PMA +PMB
Copy-number estimation using Robust Multichip Analysis (CRMA) The log-additive model: log2(PMijk) = log2ij+ log2jk + ijk sample i, SNP j, probe k. Fit using robust linear models (rlm)
100K Copy-number estimation using Robust Multichip Analysis (CRMA) Longer fragments ) less amplified by PCR ) weaker SNP signals
500K Copy-number estimation using Robust Multichip Analysis (CRMA) Longer fragments ) less amplified by PCR ) weaker SNP signals
Copy-number estimation using Robust Multichip Analysis (CRMA) Normalize to get same fragment-length effect for all hybridizations
Copy-number estimation using Robust Multichip Analysis (CRMA) Normalize to get same fragment-length effect for all hybridizations
Copy-number estimation using Robust Multichip Analysis (CRMA)